The present invention is generally in the field of biosynthesis of poly(3-hydroxyalkanoates), and more particularly to improved microbial strains useful in commercial production of polyhydroxyalkanoates.
Poly(3-hydroxyalkanoates) (PHAS) are biological polyesters synthesized by a broad range of bacteria. These polymers are biodegradable and biocompatible thermoplastic materials, produced from renewable resources, with a broad range of industrial and biomedical applications (Williams and Peoples, CHEMTECH 26:38-44 (1996)). PHA biopolymers have emerged from what was originally considered to be a single homopolymer, poly-3-hydroxybutyrate (PHB) into a broad class of polyesters with different monomer compositions and a wide range of physical properties. About 100 different monomers have been incorporated into the PHA polymers (Steinbuchel and Valentin, FEMS Microbiol. Lett. 128:219-28 (1995)).
It has been useful to divide the PHAs into two groups according to the length of their side chains and their biosynthetic pathways. Those with short side chains, such as PHB, a homopolymer of R-3-hydroxybutyric acid units, are crystalline thermoplastics, whereas PHAs with long side chains are more elastomeric. The former have been known for about seventy years (Lemoigne and Roukhelman, 1925), whereas the latter materials were discovered relatively recently (deSmet et al., J. Bacteriol. 154:870-78 (1983)). Before this designation, however, PHAs of microbial origin containing both (R)-3-hydroxybutyric acid units and longer side chain (R)-3-hydroxyacid units from C5 to C16 had been identified (Wallen and Rohweder, Environ. Sci. Technol. 8:576-79 (1974)). A number of bacteria which produce copolymers of(R)-3-hydroxybutyric acid and one or more long side chain hydroxyacid units containing from five to sixteen carbon atoms have been identified (Steinbuchel and Wiese, Appl. Microbiol. Biotechnol. 37:691-97 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 3:507-14 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 40:710-16 (1994); Abe et al., Int. J. Biol. Macromol. 16:115-19 (1994); Lee et al., Appl. Microbiol. Biotechnol. 42:901-09 (1995); Kato et al., Appl. Microbiol. Biotechnol. 45:363-70 (1996); Valentin et al., Appl. Microbiol. Biotechnol. 46:261-67 (1996); U.S. Pat. No. 4,876,331 to Doi). A combination of the two biosynthetic pathways outlined described above provide the hydroxyacid monomers. These copolymers can be referred to as PHB-co-HX (where X is a 3-hydroxyalkanoate or alkanoate or alkenoate of 6 or more carbons). A useful example of specific two-component copolymers is PHB-co-3-hydroxyhexanoate (PHB-co-3HH) (Brandl et al., Int. J. Biol. Macromol. 11:49-55 (1989); Amos and McInerey, Arch. Microbiol. 155:103-06 (1991); U.S. Pat. No. 5,292,860 to Shiotani et al.).
PHA production by many of the microorganisms in these references is not commercially useful because of the complexity of the growth medium, the lengthy fermentation processes, or the difficulty of down-stream processing of the particular bacterial strain. Genetically engineered PHA production systems with fast growing organisms such as Escherichia coli have been developed. Genetic engineering also allows for the improvement of wild type PHA production microbes to improve the production of specific copolymers or to introduce the capability to produce different PHA polymers by adding PHA biosynthetic enzymes having different substrate-specificity or even kinetic properties to the natural system. Examples of these types of systems are described in Steinbuchel and Valentin, FEMS Microbiol. Lett. 128:219-28 (1995). PCT WO 98/04713 describes methods for controlling the molecular weight using genetic engineering to control the level of the PHA synthase enzyme. Commercially useful strains, including Alcaligenes eutrophus (renamed as Ralstonia eutropha), Alcaligenes latus, Azotobacter vinlandii, and Pseudomonads, for producing PHAs are disclosed in Lee, Biotechnology and Bioengineering 49:1-14 (1996) and Braunegg et al., (1998), J. Biotechnology 65: 127-161.
The development of recombinant PHA production strains has followed two parallel paths. In one case, the strains have been developed to produce copolymers, a number of which have been produced in recombinant E. coli. These copolymers include poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-co-4HB), poly(4-hydroxybutyrate) (P4HB) and long side chain PHAs comprising 3-hydroxyoctanoate units (Madison and Huisman, 1999. Strains of E. coli containing the phb genes on a plasmid have been developed to produce P(3HB-3HV) (Slater, et al., Appl. Environ. Microbiol. 58:1089-94 (1992); Fidler and Dennis, FEMS Microbiol. Rev. 103:231-36 (1992); Rhie and Dennis, Appl. Environ. Micobiol. 61:2487-92 (1995); Zhang, H. et al., Appl. Environ. Microbiol 60:1198-205 (1994)). The production of P(4HB) and P(3HB-4HB) in E. coli is achieved by introducing genes from a metabolically unrelated pathway into a P(3HB) producer (Hein, et al., FEMS Microbiol. Lett. 153:411-18 (1997); Valentin and Dennis, J. Biotechnol. 58:33-38 (1997)). E. coli also has been engineered to produce medium short chain polyhydroxyalkanoates (msc-PHAs) by introducing the phaC1 and phaC2 gene of P. aeruginosa in a fadB::kan mutant (Langenbach, et al., FEMS Microbiol. Lett. 150:303-09 (1997); Qi, et al., FEMS Microbiol. Lett. 157:155-62 (1997)).
Although studies demonstrated that expression of the A. eutrophus PHB biosynthetic genes encoding PHB polymerase, -ketothiolase, and acetoacetyl-CoA reductase in E. coli resulted in the production of PHB (Slater, et al., J. Bacteriol. 170:4431-36 (1988); Peoples and Sinskey, J. Biol. Chem. 264:15298-303 (1989); Schubert, et al., J. Bacteriol. 170:5837-47 (1988)), these results were obtained using basic cloning plasmid vectors and the systems are unsuitable for commercial production since these strains lacked the ability to accumulate levels equivalent to the natural producers in industrial media.
For commercial production, these strains have to be made suitable for large scale fermentation in low cost industrial medium. The first report of recombinant P(3HB) production experiments in fed-batch cultures used an expensive complex medium, producing P(3HB) to 90 g/L in 42 hours using a pH-stat controlled system (Kim, et al, Biotechnol. Lett. 14:811-16 (1992)). Using stabilized plasmids derived from either medium- or high-copy-number plasmids, it was shown that E. coli XL1-Blue with the latter type plasmid is required for substantial P(3HB) accumulation (Lee, et al., Ann. N.Y. Acad. Sci. 721:43-53 (1994)). In a fed-batch fermentation on 2% glucose/LB medium, this strain produced 81% P(3HB) at a productivity of 2.1 g/L-hr (Lee, et al., J. Biotechnol. 32:203-11 (1994)). The P(3HB) productivity was reduced to 0.46 g/L-hr in minimal medium, but could be recovered by the addition of complex nitrogen sources such as yeast extract, tryptone, casamino acids, and collagen hydrolysate (Lee and Chang, Adv. Biochem. Eng. Biotechnol. 52:27-58 (1995); Lee, et al., J. Ferment. Bioeng. 79:177-80 (1995)).
Although recombinant E. coli XL 1-blue is able to synthesize substantial levels of P(3HB), growth is impaired by dramatic filamentation of the cells, especially in defined medium (Lee, et al., Biotechnol. Bioeng. 44:1337-47 (1994); Lee, Biotechnol. Lett. 16:1247-52 (1994); Wang and Lee, Appl. Environ. Microbiol. 63:4765-69 (1997)). By overexpression of FtsZ in this strain, biomass production was improved by 20% and P(3HB) levels were doubled (Lee and Lee, J. Environ. Polymer Degrad. 4:131-34 (1996)). This recombinant strain produced 104 g/L P(3HB) in defined medium corresponding to 70% of the cell dry weight. The volumetric productivity of 2 g/L-hr, however, is lower than achievable with R. eutropha. Furthermore, about 15% of the cells lost their ability to produce PHB by the end of the fermentation (Wang and Lee, Biotechnol. Bioeng. 58:325-28 (1998)).
Recombinant E. coli P(3HB-3HV) producers reportedly are unable to grow to a high density and therefore are unsuited for commercial processes (Yim, et al., Biotechnol. Bioeng. 49:495-503 (1996)). In an attempt to improve P(3HB-3HV) production in a recombinant strain, four E. coli strains (XL1-Blue, JM109, HB101, and DH5xcex1) were tested by Yim et al. All four recombinant E. coli strains synthesized P(3HB-3HV) when grown on glucose and propionate with HV fractions of 7% (Yim, et al., Biotechnol. Bioeng. 49:495-503 (1996)). Unlike other strains studied (Slater, et al., Appl. Environ. Microbiol. 58:1089-94 (1992)), recombinant XL1-Blue incorporates less than 10% HV when the propionic acid concentration is varied between 0 and 80 mM. HV incorporation and PHA formation were increased by pre-growing cells on acetate followed by glucose/propionate addition at a cell density of around 108 cells per ml. Oleate supplementation also stimulated HV incorporation. This recombinant XL1-Blue when pregrown on acetate and with oleate supplementation reached a cell density of 8 g/L, 75% of which was P(3HB-3HV) with an HV fraction of 0.16 (Yim, et al., Biotechnol. Bioeng. 49:495-503 (1996)).
One of the challenges of producing P(3HB) in recombinant organisms is the stable and constant expression of the phb genes during fermentation. Often P(3HB) production by recombinant organisms is hampered by the loss of plasmid from the majority of the bacterial population. Such stability problems may be attributed to the metabolic load exerted by the need to replicate the plasmid and synthesize P(3HB), which diverts acetyl-CoA to P(3HB) rather than to biomass. In addition, plasmid copy numbers often decrease upon continued fermentation because only a few copies provide the required antibiotic resistance or prevent cell death by maintaining parB. For these reasons, a runaway plasmid was designed to suppress the copy number of the plasmid at 30 C. and induce plasmid replication by shifting the temperature to 38 C. (Kidwell, et al., Appl. Environ. Microbiol. 61:1391-98 (1995)). Using this system, P(3HB) was produced to about 43% of the cell dry weight within 15 hours after induction with a volumetric production of 1 gram P(3HB) per liter per hour. Although this productivity is of the same order of magnitude as natural P(3HB) producers, strains harboring these parB-stabilized runaway replicons still lost the capacity to accumulate P(3HB) during prolonged fermentations.
While the instability of the phb genes in high cell-density fermentations affects the PHA cost by decreasing the cellular P(3HB) yields, the cost of the feedstock also contributes to the comparatively high price of PHAs. The most common substrate used for P(3HB) production is glucose. Consequently, E. coli and Klebsiella strains have been examined for P(3HB) formation on molasses, which cost 33-50% less than glucose (Zhang, et al., Appl. Environ. Microbiol. 60:1198-1205 (1994)). The main carbon source in molasses is sucrose. Recombinant E. coli and K. aerogenes strains carrying the phb locus on a plasmid grown in minimal medium with 6% sugarcane molasses accumulated P(3HB) to approximately 3 g/L corresponding to 45% of the cell dry weight. When the K. aerogenes was grown fed-batch in a 10 L fermenter on molasses as the sole carbon source, P(3HB) was accumulated to 70% its cell dry weight, which corresponded to 24 g/L. Although the phb plasmid in K. aerogenes was unstable, this strain shows promise as a P(3HB) producer on molasses, especially since fadR mutants incorporate 3HV up to 55% in the presence of propionate (Zhang, et al., Appl. Environ. Microbiol. 60:1198-1205 (1994)).
U.S. Pat. No. 5,334,520 to Dennis discloses the production of PHB in E. coli transformed with a plasmid containing the phbCAB genes. A recxe2x88x92, lac+ E. coli strain was grown on whey and reportedly accumulates PHB to 85% of its cell dry weight. U.S. Pat. No. 5,371,002 to Dennis et al. discloses methods to produce PHA in recombinant E. coli using a high copy number plasmid vector with phb genes in a host that expresses the acetate genes either by induction, constitutively, or from a plasmid. U.S. Pat. No. 5,512,456 to Dennis discloses a method for production and recovery of PHB from transformed E. coli strains. These E. coli strains are equipped with a vector containing the phb genes and a vector containing a lysozyme gene. High copy number plasmids or runaway replicons are used to improve productivity. The vectors are stabilized by parB or by supF/dnaB(am). Using such strains, a productivity of 1.7 g/L-hr was obtained corresponding to 46 g/L PHB in 25 hrs, after which the plasmid was increasingly lost by the microbial population. PCT WO94/21810 discloses the production of PHB in recombinant strains of E. coli and Klebsiella aerogenes with sucrose as a carbon source. PCT WO 95/21257 discloses the improved production of PHB in transformed prokaryotic hosts. Improvements in the transcription regulating sequences and ribosome binding site improve PHB formation by the plasmid based phb genes. The plasmid is stabilized by the parB locus. PHB production by this construct is doubled by including the 361 nucleotides that are found upstream of phbC in R. eutropha instead of only 78 nucleotides. It is generally believed that PHB production by recombinant microorganisms requires high levels of expression using stabilized plasmids. Since plasmids are available in the cell in multiple copies, ranging from one to several hundreds, the use of plasmids ensured the presence of multiple copies of the genes of interest. Since plasmids may be lost, stabilization functions are introduced. Such systems, which are described above, have been tested for PHB production, and the utility of these systems in industrial fermentation processes has been investigated. However, overall PHB yield is still affected by loss of phb genes.
It is therefore an object of the present invention to provide recombinant microorganisms strains useful in industrial fermentation processes which can accumulate commercially significant levels of PHB while providing stable and constant expression of the phb genes during fermentation.
It is another object of the present invention to provide transgenic microbial strains for enhanced production of poly(3-hydroxyalkanoates).
It is another object of the present invention to provide transgenic microbial strains which yield stable and constant expression of the phb genes during fermentation and accumulate commercially significant levels of PHB, and methods of use thereof.
Transgenic microbial strains are provided which contain the genes required for PHA formation integrated on the chromosome. The strains are advantageous in PHA production processes, because (1) no plasmids need to be maintained, generally obviating the required use of antibiotics or other stabilizing pressures, and (2) no plasmid loss occurs, thereby stabilizing the number of gene copies per cell throughout the fermentation process, resulting in homogeneous PHA product formation throughout the production process. Genes are integrated using standard techniques, preferably transposon mutagenesis. In a preferred embodiment wherein mutiple genes are incorporated, these are incorporated as an operon. Sequences are used to stabilize mRNA, to induce expression as a function of culture conditions (such as phosphate concentration), temperature, and stress, and to aid in selection, through the incorporation of selection-markers such as markers conferring antibiotic resistance.